1. Field of the Invention
This invention relates to the field of human dietary supplements and more specifically to improved therapeutic products prepared from yeast and containing increased selenium concentrations with reduced toxicities.
2. Background
Selenium is an essential trace element for proper physiological function in humans. Decades ago, scientists demonstrated that selenium was incorporated into the chemical structure of an enzyme called glutathione peroxidase, an enzyme that is necessary to protect erythrocyte (red blood cell) cell membranes, and other biological structures against toxic reactions with highly reactive oxygen-derived species such as peroxides, and superoxides. The role of selenium in the biochemistry of glutathione peroxidase has been studied in some detail and the most current medical information confirms that trace amounts of selenium are required to maintain normal human health. Failure to ingest and absorb the necessary amounts of selenium can lead to improper functioning of the body""s metabolic processes, and to various diseases and disorders.
Although many traditional nutrients, such as the natural occurring vitamins and minerals for which the U.S. Food and Drug Administration (FDA) has established a Recommended Daily Allowance (RDA), may be consumed in large quantities without adverse health effects, ingestion of high levels of some essential nutrients, such as certain metallic nutrients like selenium, may be toxic. To maintain ordinary health, one must balance the need for a minimum amount of such compounds with the need to protect against over-ingestion to the point of toxicity. For these compounds, ingesting low doses confers a significant nutritional benefit. However, when higher levels, i.e., amounts beyond the concentrations recognized as required for ordinary nutritional benefits, are ingested, beneficial health effects are not realized and the potential for dangerous toxicity exists.
Nutritionally beneficial quantities for daily doses for selenium have been found to be small. Nutritional selenium levels have been established by the FDA (see 21 C.F.R. 101.9(c)(8)(iv), January 1994). Humans and animals can safely metabolize limited amounts of both inorganic and organic forms of selenium and can convert non-methylated selenium to mono-ordi-or trimethylated derivatives, of which the monomethylated derivatives are most toxic. [Bedwal, R. S., et al., Medical Hypotheses, 41 (2):150-159 (August 1993)]. The FDA has adopted Reference Daily Intakes (RDIs) of 70 micrograms for selenium. Selenium dosage of 600 micrograms per day has been reported as safe. [Ferris G. M. Lloyd, et al., App. Clin. Biochem.,26:83-88 (1989)]. At about this dosage, normal activity of the enzyme glutathione reductase safely converts selenogluthatione to hydrogen selenide in the liver and erythrocytes and is ultimately excreted. Thus, at such lower dosages, the body is able to safely metabolize and excrete selenium that is present in the free metallic form.
For many years, physicians and medical researchers have studied several potential health benefits resulting from the ingestion of low levels of selenium. For example, low concentrations of sodium selenate (an inorganic form of selenium) work with vanadium to improve glucose tolerance and to increase the levels of glucose-induced insulin release. However, as with many trace elements such as selenium, at higher dosage levels or concentrations, these beneficial effects are reversed and dangerous toxicity is manifested. [Furnsinn, C. et al., Internat""l J. of Obesity and Related Metab. Dis., 19(7):458-463 (1995)].
Therefore, the administration of selenium in the natural form involves a scientific and medical trade-off because, when administered in relatively low concentrations, selenium provides beneficial health effects, however, at higher concentrations, selenium exhibits dramatic toxicity such that the potential health benefits are lost and toxicity becomes the primary concern. This trade-off is particularly problematic when selenium administration is attempted, not as a dietary supplement, but rather in the treatment of disease. However, if the toxicity problems could be overcome, increased dosages of selenium could offer substantial advances in the treatment of several important disorders that affect human health. For example, the role of selenium in maintaining the function of the enzyme glutathione peroxidase has led researchers to examine the role of selenium in several disease states. In cancer, animal studies have shown that selenium protected against chemicals and ultraviolet energy sources known to cause cancer in humans. Selenium is believed to reduce the risk of certain cancers due to its properties as a strong antioxidant. A clinical study of more than 1,300 people found that those who took a daily supplement of selenium cut their overall cancer risk by nearly 40%. [Terence Monmaney, Selenium May Fight Cancer, Study Shows, LOS ANGELES TIMES, Dec. 25, 1996, at A 1, A 29]. In addition, U.S. Pat. No. 4,599,234 teaches that a combination of a selenium species (either organic or inorganic forms) with beta-carotene and a hydroxytoluene source significantly reduced the mortality of mice that were fed carcinogens and that these effects were better than those observed for the mice that were administered either selenium or beta-carotene or hydroxytoluene. U.S. Pat. No. 4,564,634 teaches a selenium-based nutritive composition having anti-neoplastic activity in which the selenium compound is used is a novel form of selenium prepared by a reaction of selenium metal with Tung oil (9,11,13-octadecatrienoic acid). [Schrauzer, G., Inorg. and Nutr. Aspects of Cancer, p. 330 (New York: Plenum Press (1978))].
Heart disease has also been shown to be reduced in persons who consume recommended amounts of selenium in their diet. In certain studies, the levels of selenium in the blood stream have been directly correlated with the degree of progression of cardiovascular disease with those patients having the lowest levels of selenium having the most extensive coronary artery blockage. In such cases, the glutathione peroxidase enzyme is thought to exert an antioxidant effect that protects the coronary vessels from disease. In a similar mechanism, selenium is thought to interact with prostaglandins to control free radical cascades that lead to elevated levels of prostaglandins and inflammation. Patients suffering from arthritis have been shown to have low levels of plasma selenium and their clinical condition has improved with dietary supplementation of selenium.
The precise mechanism by which selenium may protect from cardiovascular disease is not known, however, free radical antioxidant xe2x80x9cscavengers,xe2x80x9d such as selenium, are believed to react with oxidants such that the oxidants are not available to form oxidized low density lipoproteins (O-LDLs). Thus, a reduction in the oxidants lowers the risk of arterial plaque deposits in blood vessels. Arterial plaque is precipitous material formed chiefly of oxidized low density lipoproteins (O-LDLs). The buildup of plaque in the form of O-LDL in the arteries is understood to be a factor in ischaemic heart disease. Free radical oxidants, many of which come from naturally occurring sources such as sun exposure, metabolism of certain nutrients, and exercise, act to oxidize low density lipoprotein (LDL) into its deleterious form, O-LDL. In contrast, high density lipoprotein (HDL) is understood to have beneficial health effects in the body. HDL is understood to be a more soluble form of lipoprotein, and its presence is not known to significantly contribute to the formation of arterial plaque. Since selenium functions to reduce the levels of O-LDL and thereby increase the level of HDL in the body, adequate quantities of selenium may decrease the likelihood of cardiovascular disease as well.
Based on the foregoing, increased concentrations of selenium are potential treatments for a variety of disorders as long as the selenium concentrations do not reach toxic levels. For this reason, several different forms of selenium have been investigated to determine the optimal form for administration to humans, either as a dietary supplement or as a therapeutic product for the treatment of disease. Yeast-derived selenium has been shown to be a less toxic form of selenium, and thus a preferred source of a selenium composition for human consumption. The selenium produced by yeast cultures undergoes a type of biosynthesis whereby inorganic selenium salts are converted to an organic form via intracellular incorporation into the yeast. These organic, biosynthesized selenium yeast derivatives are better nutritive sources of selenium because they are less toxic and more easily metabolized by the mammalian system than their inorganic counterparts. One method of producing a selenium-enriched product using food yeast such as Saccharomyces cerevisiae or Candida utilis has been reported. When dried and fed to rats, these selenium-enriched yeast effectively prevent hepatic liver necrosis. [Reed et al., Yeast Tech., AVI Publ. Co., Conn. (1973)]. Unfortunately, this method results in the production of a yeast product having a low intracellular selenium content, as well as a relatively high extracellular concentration of inorganic selenium.
Generally, high extracellular concentrations of selenium are to be avoided, while higher intracellular concentrations are preferred because this tends to indicate an increased relative concentration of selenium in the organic form which, as noted above, is preferred for administration to humans. For this reason, prior efforts at producing selenium-based yeast products have focused on the ability to provide increased intracellular concentrations of selenium. For example, U.S. Pat. No. 4,530,846 (""846) describes a method for producing a selenium-enriched yeast that yields yeast with a moderately high intracellular selenium content. The yeast produced by this method are cultivated using a procedure that involves incremental feeding of the yeast culture. With respect to the process of the ""846 Patent and the limitations on selenium concentration using that method, the ""846 patent states: xe2x80x9cWhile intracellular selenium contents of yeasts are preferably in a range of 1,000 ppm or more, even as high as 2,500 ppm, the process has, as its practical limitations, the capacity of the yeast to assimilate the selenium during the yeast growth cycle without adverse effects on yield due to the selenium additive to the nutrients.xe2x80x9d In addition to the recognized limitations on the ability to achieve higher concentrations of intracellular selenium, the prior art also demonstrates that the existing yeast-derived selenium products still exhibit substantial toxicity. For example, the LD50 for the yeast product described in the ""846 patent is reported by the assignee to be on the order of 7 mg per kilogram. In practice, the LD50 rating for a product limits the amount that may be administered to a human as part of a nutritional program or as part of an overall therapy to treat a disease. A relatively high LD50 is particularly disadvantageous when physicians or researchers attempt to administer an elevated selenium dosage, i.e., several times that recommended for dietary supplementation, in the treatment of disease.
There remains a need in the art for a yeast selenium product that provides high concentrations of selenium, preferably the organic form fixed in an intracellular form, that exhibits the lowest possible toxicities when measured by LD50. Ideally, such a product would be provided by a method to produce selenium-enriched yeast that results in: (1) a high growth rate of selenium-enriched yeast; (2) selenium-enriched yeast with high intracellular selenium content; and (3) low toxicity.
The present invention overcomes the shortcomings of the prior art by providing a method for producing a composition of highly active nutritional selenium, where (1) the selenium source of the compositions of the present invention is the natural form of biosynthesized selenium; (2) the biosynthesized selenium is entirely metabolizable by the human system, and is substantially free of toxic substances; and (3) the process can be carried out efficiently and meets requirements important for commercial production.
Therefore, an object of the present invention is to provide a method for preparing biosynthesized selenium-yeast with high selenium activity and low toxicity.
It is a further object of the present invention to provide a method for the production of biosynthesized selenium having high nutritional value and low toxicity.
Another object of the present invention is to provide an improved synthetic form of nutritional selenium that is substantially similar to the naturally occurring selenium complexes found in selenium rich foods.
It is another object of the present invention to-provide a method for the production of selenium-enriched yeast, where the source of the yeast for metabolizing selenium substrate is a yeast strain such as Saccharomyces cerevisae or Saccharomyces boulardii sequela PY31.
It is a further object of the present invention to provide a form of selenium species that is essentially non-toxic to the human body.
It is another object of the present invention to-provide for methods for the treatment of disease by administering yeast-derived selenium in quantities greater than administered for dietary supplementation.
It is a further object of the present invention to provide nutritional supplements that incorporate the selenium-enriched yeast produced by these methods.
Another object of the present invention is to provide an increased dosage of selenium that is less toxic to the human body at higher dosages such that higher dosages can be administered, if desired, including the administration of yeast selenium with chemotherapeutic agents.
The present invention relates to methods of cultivating yeast using selenium compounds resulting in a dried selenium-enriched yeast product with high biological activity, nutritional supplements comprising this dried yeast product, therapeutic products comprising this dried yeast product, uses of such dried yeast product to supplement the human diet, and methods to treat disease comprising administering dosages of selenium in combination with chemotherapeutic agents to reduce the growth of tumor cells.
The process for preparing the selenium-enriched yeast product that has a high intracellular content of organically bound trivalent selenium in a highly biologically active and non-toxic form comprises the steps of:
(1) preparing an aqueous mixture of yeast growth nutrients (aqueous media);
(2) preparing an aqueous solution of selenium salt in distilled water by dissolving the selenium salt in warm distilled water and then filtering the resulting selenium solution;
(3) adding the selenium solution to the yeast growth nutrients and mixing to form a selenium growth mixture;
(4) adding the selenium growth mixture, preferably by incremental addition, to a live yeast culture to form a selenium yeast growth solution and incubating with gentle shaking action or stirring;
(5) recovering and concentrating the yeast cells from the selenium yeast growth solution;
(6) washing the recovered yeast cells to remove extracellular selenium; and
(7) pasteurizing and/or drying the washed yeast cells.
Growth media that can be used in the firstxe2x80x94preparing step of present invention include 25xc2x0 Brix molasses [TCT, Gold Coast], 38xc2x0 Brix molasses [TCT, Gold Coast], glucose media, and potato dextrose broth. In addition, Brix molasses with a higher sugar content, e.g., 79xc2x0 Brix [TCT, Gold Coast], may be used and then diluted with distilled water to form a Brix solution of lower sugar content. Numerous other growth media that are known to support the growth of yeast from the Saccharomyces family may be used and could be readily selected by one of skill in the art. In a preferred form, a mixture of different growth media may be used.
In addition, numerous vitamins and minerals may optionally be added to the yeast growth media. Such-vitamins and minerals are selected from those known in the art to help sustain proper yeast growth, including but not limited to biotin, vitamin B1, vitamin B6, calcium pantothenoate, inositol, copper, copper sulfate, zinc, zinc sulfate, iron, and iron sulfate.
The second preparing step in the process for producing the selenium-enriched yeast of the present invention involves preparing a selenium solution by dissolving selenium salt in distilled water and filtering the resultant selenium solution. The selenium may be in the form of an amorphous solid or an organoselenium compound. In a preferred form, sodium selenite may be used. In a preferred form, a cellulose acetate filter [Corning Scientific Co.] may be used. The resultant filtered selenium solution has between about 100 ppm and 40,000 ppm selenium.
The first addition step involves adding the selenium solution to the yeast growth nutrients (aqueous media) to form a selenium growth mixture. Once the selenium solution is added, the selenium and media may be mixed by gentle shaking action or stirring for between about 1 and about 30 minutes.
In the second addition step, the selenium growth mixture is added to live yeast cells to make a selenium yeast growth solution having selenium levels between about 100 ppm and about 20,000 ppm selenium, preferably between about 200 ppm to about 10,000 ppm, and most preferably between about 250 ppm to about 1,500 ppm of selenium. The second addition step preferably involves adding the selenium growth mixture to the yeast culture incrementally. This second addition step preferably takes place under a controlled pH of from about 4.2 to about 6.0, and preferably from about 4.5 to 5.3. This second addition step also preferably takes place at a temperature from about 20xc2x0 C. to About 35xc2x0 C., and preferably about 28xc2x0 C. to about 32xc2x0 C.
The yeast employed in the second addition step preferably a food grade or edible yeast, and most preferably Saccharomyces boulardii sequela PY31. Other yeast which can be used include Saccharomyces cerevisae or Saccharomyces torula. 
As stated above, the present invention may also employ a newly isolated and purified strain of yeast, Saccharomyces boulardii sequela PY31. Specifically, Saccharomyces cerevisiae and Saccharomyces boulardii sequela are of the same genus, and Saccharomyces boulardii sequela is described as a synonym of Saccharomyces cerevisiae. More particularly, the novel yeast strain Saccharomyces boulardii sequela PY31 may be isolated from raw soil samples, and cultivated to yield quantities of yeast at a scale sufficient for developmental research and for production of commercial products. The novel strain of yeast, Saccharomyces boulardii sequela PY31, has been deposited in an International Repository in accord with the Budapest Treaty and has been assigned ATCC No. 74,366. ATCC American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209. This novel yeast strain is described in co-pending application Ser. No. 08/719,572 filed on Sep. 25, 1996. This strain may be analyzed according to the principle of cellular fatty acid analysis, to obtain a quantitative measurement of relatedness to Saccharomyces cerevisae based on their fatty acid make-up. A Euclidian Distance of less than 2.5 indicates that two yeast samples are most likely the same strain. A Euclidian Distance of less than 6.0 indicates the isolates are most likely the same subspecies and a Euclidian Distance of less than 10.0 indicates the isolates are most likely the same species. The greater the distance between the isolates, the closer the relationship taxonomically. In the present invention, the Euclidian Distance between the submitted strain (Saccharomyces boulardii PY-3-1) and LIBR S. cerevisae is 7.48, which indicates that these isolates are most likely the same subspecies, but the submitted strain is not very close to LIBR S. cerevisae due to the remote Euclidian Distance.
Specifically, the method for isolating this novel yeast strain, Saccharomyces boulardii sequela PY31, comprises:
(1) identifying a location for collection of a soil sample, which is proximal to a germanium mine (i.e., within 100 yards of a germanium mine);
(2) sampling the soil by removing about 200 g from a depth of 5 cm to 20 cm, and transporting the sample using a sterilized bag;
(3) growing the living material on three different mediums which support the growth of all yeast, and that selectively kills bacteria without killing the yeast;
(4) separating the yeast from other living matter and then repeating this process until yeast can be grown without bacterial contaminants;
(5) selecting and restreaking the yeast colonies, and repeating this process three times;
(6) selecting the yeast colonies most vital for growth in a medium enriched with germanium;
(7) growing each selected colony on malt extract agar or dextrose agar, and selecting which colonies appear most robust, and;
(8) cultivating the selected yeast by growing 1-2 slants of the yeast for about 2 days at about 30xc2x0 C. and then transferring to the cultivated yeast about 100 mL of malt extract broth and then incubating at about 30xc2x0 C. for 8-10 hours, then adding to the incubated mixture about 500 mL of malt extract broth and then growing the resulting mixture at about 30xc2x0 C. for about 6 to about 14 hours.
The present invention also teaches a use of this novel yeast strain, Saccharomyces boulardii sequela PY31, to prepare selenium-enriched, non-toxic yeast forms according to the method described herein.
As part of the second addition step, the selenium yeast growth solution is incubated to induce yeast growth. The incubation may occur with shaking or stirring at about 200 rpm for a period of about 5 hours to about 75 hours, preferably from about 15 hours to about 60 hours, and most preferably about 20 hours. This incubation occurs at a temperature of about 25xc2x0 C. to about 30xc2x0 C., and preferably about 30xc2x0 C.
The yeast cells are then isolated from the selenium yeast growth solution by centrifuging the selenium yeast growth solution, and isolating the yeast cells. In a preferred form, the centrifugation step may occur at about 3,900 rpm.
The isolated yeast cells are then washed to remove extracellular selenium. The washing step may involve washing the isolated yeast cells between 2 and 20 times with aqueous solvent, such as a buffered aqueous solution that optionally contains chelating agents such as EDTA.
Lastly, the yeast cells are pasteurized and/or dried to produce a dried yeast product. The pasteurization step may occur at between about 30xc2x0 C. and about 110xc2x0 C., preferably at about 60xc2x0 C. The resulting dried yeast product may contain from about 300 ppm to about 6,000 ppm intracellular selenium, but preferably contains more than 1000 ppm intracellular selenium, and most preferably between about 2000 ppm and about 5000 ppm.
The present invention also relates to the use of the dried selenium-enriched yeast products as dietary supplements. To prepare the yeast compositions of the invention for use as a dietary supplement, the dried yeast product is combined as the active ingredient in intimate admixture with a suitable carrier according to conventional compounding techniques. This carrier may take a wide variety of forms depending upon the form of preparation desired for administration, e.g., oral, sublingual, nasal, or parenteral.
In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed. For oral liquid preparations (e.g., suspensions, elixirs, and solutions), media containing for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used. Carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to prepare oral solids (e.g., powders, capsules, pills, and tablets). Controlled release forms may also be used. Because of their ease in administration, tablets, pills, and capsules represent advantageous oral dosage unit forms, in which cases solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques.
For parenteral products the carrier will usually comprise sterile water, although other ingredients may be included, e.g., to aid solubility or for preservation purposes. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, adjuvants, and the like may be employed.
For dietary supplementation, a composition of the present invention is generally effective when parenterally administered in amounts ranging from about 1 mg of dried yeast per dose (1 dose per body weight of about 75 kg) to about 200 mg/dose of composition. A preferred amount is from about 10 mg/dose to about 50 mg/dose, and most preferred at about 20 mg/dose. This dosage of the composition translates to an amount of from about 1 xcexcg/dose to about 200 xcexcg/dose of selenium, preferably from about 10 xcexcg/dose to about 50 xcexcg/dose of selenium, and most preferably about 20 xcexcg/dose of selenium. When orally administered, the compositions of the present invention are generally effective in approximately the same amounts as the parenteral products. Activity at this level makes the compositions particularly well suited for formulations in tablet size for oral administration. The above dosage ranges are likely to be administered at varying periods for humans, for example, from daily administration to administration at least 5 times per week. However, ultimately, the dosage regimen will depend upon the particular needs of the user. A preferred dosage regimen for dietary supplementation in humans is 1-2 doses per day.
The following examples are illustrative only and do not limit the invention in any fashion. Examples 1-3 demonstrate a process to yield a selenium yeast product having in intracellular yeast concentration of greater than 1000, greater than 1800 and approximately 2000 ppm.